Therapies designed to enhance immunity are becoming important components of cancer therapies, and could be used to increase anti-viral immunity and augment the efficacy of vaccines. Many strategies focus on biologic agents such as recombinant cytokines or monoclonal antibodies. However, the success of imiquimod and lenalidomide, the two available small molecule immune-enhancers, suggests that additional agents of this type could be useful. One reason that there are so few small-molecule immune enhancers is that there has historically been no way to conduct high-throughput screening (HTS) to find them. Imiquimod and thalidomide analogs were found to enhance immune responses by accident. We have developed an assay that should finally make it possible to screen large compound libraries for immune-enhancing small molecules. The assay is a powerful revision of an HTS that we applied successfully to the NIH's Molecular Libraries Small Molecule Repository. In the new enhanced assay, we stimulate TALL- 104 human leukemic cytotoxic T lymphocytes (CTLs) with beads coated with anti-CD3 antibodies, generating submaximal exocytosis specifically in the bead-bound population. We monitor exocytosis by measuring binding of a fluorescently-labeled antibody against LAMP-1 (CD107a) to cells using flow cytometry. This allows us to conduct a no-wash assay that can detect compounds that enhance of exocytosis and discriminate them from compounds that cause exocytosis on their own. TALL-104 CTLs serve in our strategy as both a model of an immunologically-relevant cell type as well as a surrogate for other immune cell types for which it would be difficult to devise HTS-ready assays. Conducting a screen with TALL-104 cells followed by assessment of the effects of hits on other important immune functions will likely lead to the identification of therapeutic leads or to probes that could be used to identify novel cellular targets that can be exploited to produce immune enhancement. Our three aims are designed are to 1) develop and optimize a highly sophisticated assay that will assess the effects of treating cells with compounds for different lengths of time in a single read step, then validate the optimized assay by screening the Prestwick Compound Library; 2) develop a set of secondary tests to examine the effect of hits on target cell killing by CTLs and representative functions of helper T cells, B cells and dendritic cells and 3) validate the HTS/ follow-up strategy by screening the Broad Institute's DOS informer collection of ~10K compounds. Completing our aims will provide an assay and follow-up workflow suitable for screening larger compound collections, as well as generating a number of candidate molecules obtained from screening the Prestwick and Broad collections that can be pursued in future experiments.